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Chapter 11

Chapter 11. Polymorphisms. Polymorphism. A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1% – 2% of a population. Polymorphisms can be single bases or thousands of bases. Polymorphisms may or may not have phenotypic effects.

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Chapter 11

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  1. Chapter 11 Polymorphisms

  2. Polymorphism • A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1%–2% of a population. • Polymorphisms can be single bases or thousands of bases. • Polymorphisms may or may not have phenotypic effects

  3. Polymorphic DNA Sequences • Polymorphisms are found throughout the genome. • If the location of a polymorphic sequence is known, it can serve as a landmark or marker for locating other genes or genetics regions. • Each polymorphic marker has different versions, or alleles.

  4. Types of Polymorphic DNA Sequences • RFLP: restriction fragment length polymorphisms • VNTR: variable number tandem repeats (8 to >50 base pairs) • STR: short tandem repeats (1–8 base pairs) • SNP: single-nucleotide polymorphisms

  5. Types of Polymorphisms • Single nucleotide polymorphisms (SNPs) • A Single Nucleotide Polymorphism, or SNP (pronounced "snip"), is a small genetic change, or variation, that can occur within a person's DNA sequence. • Common SNPs are defined as >1% in at least one population • Rare SNPs are hard to identify and validate • But, it is estimated that there are a large number per individual • Some areas more variable than others (HLA)

  6. GTCCAGTCTAGC GAATTC GTGGCAAAGGCT CAGGTCAGATCG CTTAAG CACCGTTTCCGA GTCCAGTCTAGC GAA A TC CG TGGC C A AGGCT GC G CAGGTCAGATCG CTT ACCG T TCCGA T AG Point mutations AAAGGCT GTCCAGTCTAGC GA AGCGA ATTC GTGGC TTTCCGA CAGGTCAGATCG CT TCGCT TAAG CACCG Insertion GTTCTAGC GAATTC GTGGC AAA GGCT GAATTC GTGG TCAGATCG CTTAAG CACCG TTT CCGA CTTAAG CACC GTTCTAGC GAATTC GTGGC AAAAAA GGCT GAATTC GTGG TCAGATCG CTTAAG CACCG TTTTT T CCGA CTTAAG CACC Fragment insertion (or deletion) Restriction Fragment Length Polymorphisms • Restriction fragment sizes are altered by changes in or between enzyme recognition sites.

  7. RFLP • RFLP (often pronounced "rif lip”) is used to follow a particular sequence of DNA as it is passed on to other cells. • RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. RFLPs can be used determine the disease status of an individual. RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in centiMorgans.

  8. RFLP • An RFLP is a sequence of DNA that has a restriction site on each end with a "target" sequence in between. • A target sequence is any segment of DNA that bind to a probe by forming complementary base pairs. • A probe is a sequence of single-stranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be detected. When a probe base pairs to its target, the investigator can detect this binding and know where the target sequence is since the probe is detectable.

  9. RFLP • RFLP produces a series of bands when a Southern blot is performed with a particular combination of restriction enzyme and probe sequence

  10. RFLP • Recall: EcoR I binds to its recognition sequence GAATTC and cuts the double-stranded DNA as shown EcoRI -GAATTC- -CTTAAG-

  11. In the segment of DNA shown below, you can see the elements of an RFLP; a target sequence flanked by a pair of restriction sites. When this segment of DNA is cut by EcoR I, three restriction fragments are produced, but only one contains the target sequence which can be bound by the complementary probe sequence (purple).

  12. Restriction Fragment Length Polymorphisms • RFLP genotypes are inherited. • For each locus, one allele is inherited from each parent. Southern blot band patterns

  13. Let's look at two people and the segments of DNA they carry that contain this RFLP (for clarity, we will only see one of the two stands of DNA). Since Jack and Jill are both diploid organisms, they have two copies of this RFLP. When we examine one copy from Jack and one copy from Jill, we see that they are identical: • Jack 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC- • Jill 1: -GAATTC---(8.2 kb)---GCATGCATGCATGCATGCAT---(4.2 kb)---GAATTC-

  14. When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of the target sequence (difference in italics). • Jack 2: -GAATTC--(1.8 kb)-CCCTTT--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC-Jill 2: -GAATTC--(1.8 kb)-GAATTC--(1.2 kb)--GCATGCATGCATGCATGCAT--(1.3 kb)-GAATTC-

  15. Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will have one band in common and one band that does not match the other's in molecular weight:

  16. Parentage Testing by RFLP • Which alleged father’s genotype has the paternal alleles? AF2 Mother AF 1 Locus Locus Locus 1 2 1 2 1 2 Child Locus 1 2

  17. M S1 S2 V E M M S1 S2 V E M M S1 S2 V E M Evidence Testing by RFLP • Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, M = molecular weight standard) Locus 1 Locus 2 Locus 3

  18. Short Tandem Repeat Polymorphisms (STR) • STRs are repeats of nucleotide sequences. • AAAAAA…: mononucleotide • ATATAT…: dinucleotide • TAGTAGTAG…: trinucleotide • TAGTTAGTTAGT…: tetranucleotide • TAGGCTAGGCTAGGC…: pentanucleotide • Different alleles contain different numbers of repeats. • TTCTTCTTCTTC : four-repeat allele • TTCTTCTTCTTCTTC: five-repeat allele

  19. GTTCTAGC GGCC GTGGC AGCTAGCTAGCT GCTG GGCC GTGG CAAGATCG CCGG CACCG TCGATCGATCGA CGAC CCGG CACC Short Tandem Repeat Polymorphisms • STR alleles can be analyzed by fragment size (Southern blot). One repeat unit Allele M 1 2 M Restriction site Allele 1 GTTCTAGC GGCC GTGGC AGCTAGCTAGCTAGCT GCTG GGCC GTGG CAAGATCG CCGG CACCG TCGATCGATCGATCGA CGAC CCGG CACC tandem repeat Allele 2

  20. Short Tandem Repeat Polymorphisms • STR alleles can also be analyzed by amplicon size (PCR). Primers (Genotype)

  21. Short Tandem Repeat Polymorphisms • Allelic ladders are standards representing all alleles observed in a population. 11 repeats 5 repeats (Allelic ladder) Genotype: 6,8 Genotype: 7,9

  22. Short Tandem Repeat Polymorphisms • Multiple loci are genotyped in the same reaction using multiplex PCR. • Allelic ladders must not overlap in the same reaction.

  23. FGA TPOX D8S1179 vWA PentaE D18S51 D2S11 THO1 D3S1358 Short Tandem Repeat Polymorphisms by Multiplex PCR

  24. AmelogeninLocus, HUMAMEL • The amelogenin locus is not an STR. • The HUMAMEL gene codes for amelogenin-like protein. • The gene is located at Xp22.1–22.3 and Y. • X allele = 212 bp • Y allele = 218 bp • Females (X, X): homozygous • Males (X, Y): heterozygous

  25. 11 repeats 5 repeats 11 repeats 5 repeats (Allelic ladder) Analysis of Short Tandem Repeat Polymorphisms by PCR • STR genotypes are analyzed using gel or capillary gel electrophoresis. Genotype: 7,9

  26. Child’s alleles Mother’s alleles Father’s alleles STR-PCR • STR genotypes are inherited. • One allele is inherited from each parent.

  27. Which alleged father’s genotype has the paternal alleles? Parentage Testing by STR-PCR

  28. Evidence Testing by STR-PCR • Which suspect—S1 or S2—was at the crime scene? (V = victim, E = crime scene evidence, AL = allelic ladder) AL S1 S2 V E M AL S1 S2 V E M AL S1 S2 V E M Locus 1 Locus 2 Locus 3

  29. Short Tandem Repeat Polymorphisms: Y-STR • The Y chromosome is inherited in a block without recombination. • STR on the Y chromosome are inherited paternally as a haplotype. • Y haplotypes are used for exclusion and paternal lineage analysis.

  30. Short Tandem Repeat Polymorphisms: mini-STR • Mini-STRs are STRs on smaller amplicons. • Recommended for degraded specimens • Used to identify remains from mass graves and disaster areas

  31. MHC • Major histocompatibilitycomplex (MHC) is a cell surface molecule encoded by a large gene family in all vertebrates. MHC molecules mediate interactions of leukocytes. MHC determines compatibility of donors for organ transplant as well as one's susceptibility to an autoimmune disease via crossreacting immunization. In humans, MHC is also called human leukocyte antigen (HLA). • Protein molecules—either of the host's own phenotypeor of other biologic entities—are continually synthesized and degraded in a cell. Occurring on the cell surface, each MHC molecule displays a molecular fraction, called an epitope. The presented antigen can be either self or nonself. • The MHC gene family is divided into three subgroups—class I, class II, and class III. • We will discuss these later…..

  32. SNPs & Function: We know so little.. • Majority are “silent”: No known functional change • Alter gene expression/regulation • Promoter/enhancer/silencer • mRNA stability • Small RNAs • Alter function of gene product • Change sequence of protein

  33. Large and Small Scale Polymorphisms • Copy Number of Polymorphisms • Regional “repeat” of sequence • 10s to 100s kb of sequence • Estimate of >10% of human genome • Multi-copy in many individuals • Duplicons • 90-100% similarity for >1 kb • 5-10% of genome (5% exons elsewhere) • Multi-copy (high N) in all individuals

  34. Copy Number Variation • Across the Genome • Frequency of CNVs • Most are uncommon (<5%) • Familial vs Unrelated Studies • Associated with Disease • OPN1LW Red/green colorblind • CCL3L1 Reduced HIV Infection • CYP2A6 Altered nicotine metabolism • VKORC1 Warfarin metabolism

  35. Engraftment Testing Using STR • Allogeneic bone marrow transplants are monitored using STR. Recipient receives his or her own purged cells. Recipient receives donor cells. Autologous transplant Allogeneic transplant A recipient with donor marrow is a chimera.

  36. Engraftment Testing Using STR • There are two parts to chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis. Before transplant Donor Recipient After transplant Complete (full Mixed Graft failure chimerism) chimerism

  37. Locus: 1 2 3 4 5 Engraftment Testing Using STR:Informative Analysis • STR are scanned to find informative loci (donor alleles differ from recipient alleles). • Which loci are informative?

  38. Engraftment Testing Using STR:Informative Analysis • There are different degrees of informativity. • With the most informative loci, recipient bands or peaks do not overlap stutter in donor bands or peaks. • Stutter is a technical artifact of the PCR reaction in which a minor product of n-1 repeat units is produced.

  39. Examples of Informative Loci (Type 5) [Thiede et al. Leukemia 2004;18:248] Recipient Stutter Donor Recipient Donor

  40. Examples of Non-informative Loci (Type 1) Recipient Donor

  41. vWA TH01 Amel TPOX CSF1PO Engraftment Testing Using STR:Informative Analysis • Which loci are informative?

  42. A(R) + A(D) A(R) A(D) Engraftment Testing Using STR:Engraftment Analysis • Using informative loci, peak areas are determined in fluorescence units or from densitometry scans of gel bands. • A(R) = area under recipient-specific peaks • A(D) = area under donor-specific peaks

  43. Engraftment Testing Using STR:Engraftment Analysis • Formula for calculation of % recipient or % donor (no shared alleles). A(R) % Recipient DNA = × 100 A(R) + A(D) A(D) % Donor DNA = × 100 A(R) + A(D)

  44. Engraftment Testing Using STR:Engraftment Analysis • Calculate % recipient DNA in post-1 and post-2: Use the area under these peaks to calculate percentages.

  45. Engraftment Analysis of Cellular Subsets • Cell subsets (T cells, granulocytes, NK cells, etc.) engraft with different kinetics. • Analysis of cellular subsets provides a more detailed description of the engrafting cell population. • Analysis of cellular subsets also increases the sensitivity of the engraftment assay.

  46. Engraftment Analysis of Cellular Subsets • T cells (CD3), NK cells (CD56), granulocytes, myeloid cells (CD13, CD33), myelomonocytic cells (CD14), B cells (CD19), stem cells (CD34) • Methods • Flow cytometric sorting • Immunomagnetic cell sorting • Immunohistochemistry + XY FISH

  47. Engraftment Analysis of Cellular Subsets • Detection of different levels of engraftment in cellular subsets is split chimerism. R D T G R = recipient alleles D = donor alleles T = T-cell subset (mostly recipient)G = granulocyte subset (mostly donor)

  48. Single Nucleotide Polymorphisms (SNPs) • Single-nucleotide differences between DNA sequences. • One SNP occurs approximately every 1250 base pairs in human DNA. • SNPs are detected by sequencing, melt curve analysis, or other methods. • 99% have no biological effect; 60,000 are within genes.

  49. SNP Detection by Sequencing T/T T/A A/A 5′AGTCTG 5′AG(T/A)CTG 5′AGACTG

  50. SNP Haplotypes • SNPs are inherited in blocks or haplotypes.

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